High quality circular genome sequences were generated for the Clostridium
chauvoei type strain DSM 7528T (ATCC 10092T) and a field strain 12S0467
isolated in Germany. Comparative genome analysis of the two strains revealed
few inversions and translocations in local collinear blocks, indicating a
conserved genome with only a small number of accessory genes. Significant
homology for C. chauvoei was observed with C. septicum. The species genome
shows a large number of genes, the products of which are involved in
proteolysis, cleavage of glycosidic linkages (sialidases) and metal ion
transportation. Triplicates of fliC were identified in each of the two
circular genomes. Sporulation and germination process related genes were
homologous to those of the Clostridia cluster I species with highest homology
to C. septicum, but novel variations in regulatory genes were also identified.
A comparative genomic study was carried out with a total of 64 C. chauvoei
strains. The strain collection mostly included strains of European origin
whereas few strains were of exotic origin. Pan-genome analysis of the species
based on the available C. chauvoei strains shows that the species has an open
pan-genome structure. New gene acquisition was observed to be very limited.
This probably indicates that the species is undergoing replication only in
very isolated areas e.g. inside the host tissue, so that the chances of
acquiring foreign genetic material are limited. The predicted prophage
identified in the genomes was similar for all strains, indicating that the
genomes are immune to any new phage type. A CRISPR type I-B system was
identified in all genomes which may contribute to comprehensive phage
immunity. There was an obvious correlation of strains from the same
region/farm i.e. displaying the same repeat numbers/spacer sequences. The
strains originating from outside Europe showed a unique spacer matrix
composition. Homologous recombination plays an important role in the evolution
of some bacterial pathogens. This study found only a limited number of
possible recombination events contributing towards the evolution of C.
chauvoei. Maximum Likelihood phylogenetic analysis based on the core genome
(Parsnp) identified associated isolates with strong bootstrap support values
(100%). These values were highly prominent for strains originating from the
same farm/outbreak/animal. Reference genome and alignment free analysis
methods (kSNP 3) based on pan-genome SNPs (SNP, Single Nucleotide
Polymorphism) were found to be useful for initial clustering of isolates. The
strains were clustered based on unique SNPs. The number of allele specific
SNPs which are unique for each strain was higher for strains from exotic
origin. Read mapping and SNP calling (Snippy) based on a related reference
genome (12S0467) was able to provide novel insights to understand the general,
outbreak and within-host microevolution of a pathogen. The median pairwise SNP
difference value was higher for C. chauvoei strains from Bavaria and Austria
as compared to strains from Lower Saxony. The diversity of geographically
related strains could therefore be indicative of other environmental factors
or herd management influencing strain microevolution. Nonsynonymous SNPs
contributed significantly to the observed microevolution of the pathogen.
Based on the strain variability observed for strains from the same host and
strains from the same outbreak, this study also proved the possibility of
genetically different C. chauvoei populations in one host. A very recent
study, based on genome sequence data, has pointed out the applicability of
strain differentiation based on CRISPR spacer sequences for C. chauvoei. The
current study similarly proved the applicability of typing approaches for this
pathogen based on CRISPR elements and core genome MLST. The CRISPR spacer
diversity was found to be inadequate to differentiate strains of European
origin. However core genome MLST was able to differentiate the C. chauvoei
strains within Europe and Germany and thus proved to be a valuable tool for
strain differentiation. The current study applied the advantage of long read
based sequencing technology to generate high quality complete genome reference
sequences for a field strain from Germany and the type strain of C. chauvoei.
The phylogenetic positioning of the species within the genus was evaluated and
the close relatedness to C. septicum was confirmed. Several genome analysis
software tools provided novel insights in the genome content and composition
of the pathogen. Comparative genome sequence analysis based on 64 strains
showed limited horizontal gene transfer and novel gene acquisition. Strain
typing based on core genome MLST analysed used the first time for this species
could differentiate strains even at farm level.